Recombinant reverse transcriptases

ABSTRACT

The present invention relates to a gene that encodes a hyperactive reverse transcriptase having DNA polymerase activity and substantially reduced RNase H activity, vectors containing the gene and host cells transformed with the invention. The present invention also includes a method of producing the hyperactive reverse transcriptase, producing cDNA from mRNA using the reverse transcriptase of the invention, kits and assay templates made using the hyperactive reverse transcriptase.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to recombinant Reverse Transcriptase (RT)enzymes with modified activity, and more particularly, to selectivelymutated RTs with enhanced RNA directed, DNA polymerase activity thatproduce longer cDNAs, higher aRNA yields.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with the use of RT enzymes for use in a wide variety ofassays used by molecular biologists, as an example.

Heretofore, in this field, the RNA directed, DNA polymerase activityresponsible for the synthesis of complementary DNA (cDNA) from an RNAtemplate has been accomplished using reverse transcriptase enzymes,whether purified or recombinant. One such use for RTs if fortranscription-based amplification systems, e.g., amplifying RNA and DNAtarget sequences.

Transcription-based amplification methods find use in a wide variety ofsettings, e.g., routine clinical laboratory use in diagnostic tests suchas direct detection of pathogens. Another such use for RTs is in theinitial step for RT-PCR (polymerase chain reaction) used to amplify anRNA target for analysis and/or cloning. In RT-PCR, the RT is used tomake an initial complementary DNA (cDNA) copy of the RNA target, whichis then amplified by successive rounds of DNA replication.

RTs have three primary enzymatic activities: a RNA-directed, DNApolymerase activity; a DNA-directed, DNA polymerase activity; and anRNase H activity. The RNase H activity degrades specifically RNA foundin an RNA:DNA duplex. An initial goal of many molecular biologists wasto identify an RT that had no detectable RNase H activity while stillmaintaining DNA polymerase activity. An RT having no RNase H activitywould finds particular use because degradation of the RNA strand ofRNA:DNA intermediates by RNase H causes unwanted degradation of thetemplate reducing cDNA yields. U.S. Pat. Nos. 5,244,797, 5,540,776,5,668,005, 6,063,608, 6,589,768 and 6,610,522, disclose one such mutantRT, wherein a gross deletion mutant with no detectable RNase H activityis taught.

U.S. Pat. No. 5,998,195 teaches a method of reducing the level of RNaseactivity in an RT preparation by using an expression vector or plasmidcontaining a cloned version of the gene for an MMLV-RT which, when usedto transform a suitable host cell such as E. coli, leads to theexpression of the gene and the generation of a gene product having theDNA- and RNA-directed DNA polymerase activities and RNase H activityassociated with retroviral reverse transcriptases.

A host cell with a reduced level of ribonuclease activity as compared towild-type strains is used to provide a source of RT that has endogenouslevels of RNase activity below that of previous recombinantpreparations.

Yet others have purified RT, e.g., Goff et al., U.S. Pat. No. 4,943,531(1990) and Kotewicz et al., U.S. Pat. No. 5,017,492, which havedescribed methods for the purification of reverse transcriptase derivedfrom Moloney Murine Leukemia Virus (MMLV-RT) and expressed in E. coli.These expression constructs and isolation and purification methods formthe basis for the majority of commercial reverse transcriptasepreparations.

SUMMARY OF THE INVENTION

The present invention relates to a gene that encodes a hyperactivereverse transcriptase having enhanced DNA polymerase activity. IncreasedDNA polymerase activity is achieved by one or more point mutations inthe DNA processivity domain of the RT. Using the mutant RTs of thepresent invention greatly enhanced yields of aRNA may be achieved fromtemplate amounts in picogram amounts. In addition to enhancedamplification, the hyperactive RTs were found to consistently produceextra-long cDNAs, that is, messages exceeding 9 kb.

The present invention may also include one or more mutations to thenucleotide selection domain, which is located near the amino terminus ofthe RT. The present invention may also include one or more mutations inthe processivity domain, which facilitates the formation of longer cDNAproducts. Mutants in the processivity domain of RT also exhibitsubstantially reduced RNase H activity (e.g., between about 0.1, 0.5,1.0, 2.5, 5.0, 10 to about 50% percent of the wild-type activity of MMLVRT). The hyperactive mutants described herein are able to produce,enhanced amplification of mRNA to cDNA from very small quantities oftemplate in both single and double rounds of amplification whilemaintaining message ratio fidelity.

More particularly, the present invention includes an isolatedhyperactive reverse transcriptase that includes one or more pointmutations in the processivity domain and/or one or more point mutationsin the nucleotide selection domain. The reverse transcriptase may be,e.g., an AMV, M-MLV, HTLV-1, BLV, RSV, HFV, R2 Bombyx mori or HIVreverse transcriptase. The hyperactive reverse transcriptase is encodedby a modified nucleotide sequence that encodes a modified amino acidsequence modified in the processivity domain generally correspond withamino acids 497 to 671 of M-MLV reverse transcriptase. The modificationsto the nucleotide selection domain may correspond to amino acids 153 to158 of M-MLV reverse transcriptase. As disclosed herein, the reversetranscriptase may be used in the preparation of full-length cDNA and maybe a hyperactive reverse transcriptase that is produced recombinantlyand purified to, e.g., greater than about 90% pure.

Examples of mutations that have been developed and that show an increasein activity over the wild-type enzyme, as described and characterizedherein include, e.g., mutations in the processivity domain with one ormore of the following mutations corresponding to the amino acids inMMLV-RT: H638G, Y586A, D653N, D524N, D524E and E562D. For mutations inthe nucleotide selection domain these may include one or more of thefollowing mutations corresponding to the amino acids in MMLV-RT: F155,D153, A154, F155, F156, C157, or L158. In one group of specific examplesthat demonstrate the structure and functional relationship between themutations, the mutation in the processivity domain may include one ormore of the following mutations corresponding to the amino acids inMMLV-RT: H638G, Y586A, D653N, D524N, D524E and E562D and the mutation inthe nucleotide selection domain may include one or more of the followingmutations corresponding to the amino acids in MMLV-RT: F155Y. Thehyperactive reverse transcriptase produces a yield of greater than about1, 5, 7, 10, 15 or about 25 ug of an aRNA from 100 ng of template RNA ina single amplification reaction. Alternatively, the hyperactive reversetranscriptase produces a yield of greater than about 1, 2, 5 or even 10ug of an aRNA from 10 pg of template RNA after a two-round amplificationreaction. The hyperactive reverse transcriptase may produces a cDNAgreater than about 6, 9 or even 11 or from between about 6 to about 15kilobases, or greater than 15 kilobases in a single cDNA synthesisreaction. The hyperactive reverse transcriptase has a DNA polymeraseactivity of greater than about 200 Units per microgram, e.g., betweenabout 0.1 and 300 Units per microgram. Generally, the reversetranscriptase of the present invention has an RNase H activity ofbetween about 0.1 and about 25 percent of the wild-type RNase H activityof reverse transcriptases.

The present invention also includes an isolated reverse transcriptasehaving substantially reduced RNase H activity that one or more pointmutations in the processivity domain. The RNase I activity of thereverse transcriptase has between about 0.1 and 50% of wild-typeactivity or between about 1 and 10% of wild-type RNase H activity of awild-type reverse transcriptase, e.g., an MMLV-RT. The mutation in theprocessivity domain may include one or more of the following mutationscorresponding to the amino acids in MMLV-RT: H638G, Y586A, D653N, D524N,D524E and E562D and may further include a mutation in the nucleotideselection domain comprises a mutation of residue F155 in MMLV-RT. Thereverse trancriptase may be isolated and purified and include one ormore mutations in the processivity domain corresponding to the aminoacids in MMLV-RT: H638G, Y586A, D653N, D524N, D524E and E562D and amutation in the nucleotide selection domain at F155Y.

Yet another embodiment of the present invention is an isolated proteinhaving DNA polymerase activity and substantially reduced RNase Hactivity comprising one or more mutations in the processivity domain andone or more mutations in the nucleotide selection domain. The isolatedprotein having DNA polymerase activity and substantially reduced RNase Hactivity may produces a yield of greater than about 1, 5, 7, 10, 12, 15,25 ug of an aRNA from 100 ng of template RNA in a single amplificationreaction. Another method for characterizing the activity of the reversetranscriptase enzyme mutants disclosed herein is that the reversetranscriptase protein produces an aRNA yield of greater than about 20%as compared to an equivalent wild-type Reverse Transcriptase enzyme.Another characteristic is that the reverse transcriptase proteinproduces a yield of greater than about 1, 5 or 10 ug of an aRNA from 10pg of template RNA after a two-round amplification reaction; a cDNAgreater than about 6, 9 or even 11 kilobases in a single cDNA synthesisreaction; a cDNA greater than about 6 to about 15 kilobases in a singlecDNA synthesis reaction or even a cDNA greater than about 15 kilobasesin a single cDNA synthesis reaction. Generally, the DNA polymeraseactivity is greater than about 200 Units per microgram, e.g., betweenabout 0.1 and 300 Units per microgram. Functionally, the mutant reversetranscriptase will have between about 0.1 and about 25 percent of thewild-type RNase H activity.

The present invention also includes an isolated and purified reversetranscriptase protein comprising one or more mutations in the nucleotideselection domain and may be selected from, e.g., AMV, M-MLV, HTLV-1,BLV, RSV, HFV, R2 Bombyx mori and/or HIV reverse transcriptase. Thereverse transcriptase may also be modified at the nucleotide sequence toencodes a modified amino acid sequence in the processivity domaincorresponding to amino acids 497 to 671 of M-MLV reverse transcriptase.When the nucleotide selection domain is mutated this may be one or morepoint mutations in the nucleotide selection domain corresponding toamino acids 153 to 158 of M-MLV reverse transcriptase and may be used inthe preparation of full-length cDNA.

A process for making a protein with hyperactive reverse transcriptaseactivity may include the steps of: transforming a host cell with thehyperactive RT comprising a mutation in the processivity domain thatcomprises one or more of the following mutations corresponding to theamino acids in MMLV-RT: H638G, Y586A, D653N, D524N, D524E and E562D andfurther comprising a F155Y mutation in the nucleotide selection domainof MMLV-RT and culturing the host cell under conditions such that thehyperactive reverse transcriptase is produced by the host cell.

The present invention also includes an isolated and purified nucleicacid encoding a hyperactive reverse transcriptase with a mutation in theprocessivity domain and/or in the nucleotide selection domain. Forexample, the nucleic acid sequence may be modified to encode ahyperactive reverse transcriptase having a mutation that corresponds toand includes, e.g., an H638G mutation of the MMLV-RT, an F155Y mutationor an F155Y mutation and an H638G mutation. The nucleic acid has SEQ IDNo.: 1 and further include, e.g., a nucleic acid segment encoding aleader sequence and/or encode a protein segment other than thehyperactive reverse transcriptase to form, e.g., a fusion protein.Another embodiment of the present invention is a vector that includes anucleic acid having a nucleic acid encoding a hyperactive reversetranscriptase that encodes a mutation in the processivity domain and/orin the nucleotide selection domain.

Yet another embodiment of the present invention is a host celltransformed with an expression vector having a nucleic acid encoding anamino acid of SEQ ID NO.: 2, for a hyperactive reverse transcriptase.The host cell may be a bacteria, fungi, plant, or even a mammalian cell.One example of a host is E. coli or even P. pastoris. The host cell mayeven be transformed to express a hyperactive reverse transcriptase. Thehost cell, vector and constructs disclosed herein may be used in aprocess for making an isolated hyperactive reverse transcriptase thatincludes the steps of transforming a host cell with an isolated nucleicacid that encodes a hyperactive reverse transcriptase; and culturing thehost cell under conditions such that the hyperactive reversetranscriptase is produced.

The hyperactive reverse transcriptase may include one or more mutationsreplace at least one of the amino acids of the processivity domain andthe nucleotide selection domain, with an alternative naturally occurringL-amino acid, the replacement being selected from the group consistingof: (1) a substitution of any of isoleucine, valine, and leucine for anyother of these amino acids; (2) a substitution of aspartic acid forglutamic acid or vice versa; (3) a substitution of glutamine forasparagine or vice versa; (4) a substitution of serine for threonine orvice versa; (5) a substitution of glycine for alanine or vice versa; (6)a substitution of alanine for valine or vice versa; (7) a substitutionof methionine for any of leucine, isoleucine, or valine and vice versa;and (8) a substitution of lysine for arginine or vice versa.Alternatively, the replacement may be selected from the group consistingof: (1) a substitution of any of isoleucine, valine, or leucine for anyother of these amino acids; (2) a substitution of aspartic acid forglutamic acid or vice versa; (3) a substitution of glutamine forasparagine or vice versa; and (4) a substitution of serine for threonineor vice versa and wherein the hyperactive reverse transcriptasecomprises a hyperactive reverse transcriptase.

The present invention also includes a variety of kits that use thepresent invention, which will generally include instructions for the useof the hyperactive reverse transcriptase and a variety of buffers,controls and the like. One example of a kit may be used to synthesizenucleic acid synthesis, and includes in a suitable container: ahyperactive reverse transcriptase; and a reaction solution for thereverse transcriptase. The kit may also include information insert mayinclude information for using the reverse transcriptase, a reactionsolution comprises a 10× concentrated reverse transcriptase reactionbuffer, a primer, a reverse transcriptase buffer, a PCR buffer, a singlecontained with a mix of nucleotides or containers that each holdindividual nucleotides, a buffer for in vitro transcription, a templatepurification column and/or one or more magnetic particles suitable fornucleic acid purification. Alternatively, the kit for nucleic acidsynthesis, may include in a suitable container a hyperactive reversetranscriptase comprising one point mutation in the processivity domain;and a reaction solution for the reverse transcriptase. Another kit mayincludes suitable containers having a hyperactive reverse transcriptasecomprising one point mutation in the processivity domain and one pointmutation in the nucleotide selection domain; and a reaction solution forthe reverse transcriptase.

The present invention also includes a method for RNA amplification thatincludes the steps of, reverse transcribing an RNA template into asingle-stranded cDNA with a hyperactive reverse transcriptase in thepresence of an oligonucleotide comprising a transcriptional promoter anda primer, purifying the single-stranded cDNA; and generating amplifiedRNA (aRNA) using an RNA polymerase. Alternatively, a method for RNAamplification may includes the steps of: reverse transcribing an RNAtemplate into a single-stranded cDNA with a hyperactive reversetranscriptase in the presence of an oligonucleotide comprising atranscriptional promoter and a primer, converting the single-strandedcDNA into double-stranded cDNA using a DNA polymerase, purifying thedouble-stranded cDNA and generating amplified RNA (aRNA) using an RNApolymerase. The method may also include purifying the aRNA and aRNA madeusing the methods disclosed herein.

Yet another kit may be for RNA amplification and includes in one or moresuitable containers a hyperactive reverse transcriptase that includesone or more point mutations in the processivity domain and one or morepoint mutations in the nucleotide selection domain; an oligonucleotidewith a transcriptional promoter region and/or oligo(dT) region; a DNApolymerase; and an RNA polymerase. The kit may also includes one or moreof the following: an insert may be provided that includes informationfor using the optimized reverse transcriptase, a 10× concentratedreverse transcriptase reaction buffer, a primer, a reverse transcriptasebuffer, a DNA Polymerase buffer, a mix of nucleotides, separatecontainers for individual nucleotides, a buffer for in vitrotranscription, a nucleic acid purification column and/or a magneticparticle or particles suitable for nucleic acid purification.

Another kit for RNA amplification may include one or more suitablecontainers that include: a hyperactive reverse transcriptase with one ormore point mutations in the processivity domain; an oligonucleotide witha transcriptional promoter region and oligo(dT) region; a DNApolymerase; and an RNA polymerase. The kits, methods and compositionsdisclosed herein may be used to make an aRNA including a ssDNA or aDNA:RNA hybrid made from an RNA template by a hyperactive reversetranscriptase. Also included may be an RT-PCR kit with one or moresuitable containers: a hyperactive reverse transcriptase, two or moreprimers, nucleotides, a thermostable DNA polymerase and an RT-PCTbuffer. The same container or a separate container may also be providedthat includes one or more reverse transcriptases in addition to thehyperactive reverse transcriptase of the present invention as a controlor to provide additional reverse transcriptase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures in which correspondingnumerals in the different figures refer to corresponding parts and inwhich:

FIG. 1 shows the domain structure of MMLV RT, point mutations relevantherein are marked;

FIG. 2 shows the gene sequence of F155Y;H638G MMLV RT (SEQ ID NO: 1);

FIG. 3 shows the protein sequence of F155Y;H638G MMLV RT (SEQ ID NO: 2);

FIG. 4 is a gel that shows a comparison of cDNA Product Lengths byVarious Mutant MMLV RT Enzymes using RNA Templates from 0.5 to 9.0 kb inSize;

FIG. 5 is a gel of the cDNA synthesis products using a 9 kb RNA templatewith MMLV RT mutants in the presence of MgCl₂ or MnCl₂;

FIG. 6 is a graph that demonstrates single round RNA Amplification with100 ng Rat Thymus Total RNA: MMLV RT Mutant Comparisons., the aRNAyields were determined by UV absorbance at 260 nm, samples wereperformed in duplicate;

FIG. 7 is a graph of a single round RNA Amplification with 90 ng HeLa S3Total RNA: MMLV RT Mutant Comparisons (the aRNA yields were determinedby UV absorbance at 260 nm, performed in duplicate); FH=F155Y;H638G MMLVRT; SSII=SuperScript II; MMLV=wild-type MMLV RT; AMV=AvianMyeloblastosis Virus RT;

FIG. 8 is a graph that demonstrates the yield from a two round RNAAmplification with 10-1000 pg HeLa S-3 Total RNA: MMLV RT MutantComparisons (AMV=Avian Myeloblastosis Virus RT; FYHG=F155Y;H638G MMLVRT; SSII=SuperScript II; The aRNA yields were determined by UVabsorbance at 260 nm, performed in duplicate);

FIG. 9 is a graph that demonstrates the yield from Two Round RNAAmplification Comparing F155Y;H638G MMLV RT and AMV RT (input total RNAwas HeLa-S3, at 1 ng and 10 pg);

FIG. 10 is a graph that shows compares a hyperactive reversetranscriptase with the Standard Affymetrix aRNA Protocol Comparison toMessageAMP containing F155Y;H638G MMLV RT; and

FIG. 11 is a graph that shows a Scatter plot comparing F155Y;H638G MMLVRT (x axis) vs. SSII (y axis) Signal Intensities from a Human FocusArray.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts whichcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

DEFINITIONS

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not limit the invention, except as outlined in the claims. As usedthroughout the present specification the following abbreviations areused: kb, kilobase (pairs); kD, kilodalton; PCR, polymerase chainreaction; RT, reverse transcriptase; MMLV, murine moloney leukemiavirus; AMV, avian myoblastosis virus; RSV, Rous sarcoma virus; HIV,human immunodeficiency virus; HFV, human foamy virus.

The invention relates to the production of hyperactive RTs. The presentinvention also includes RTs having DNA polymerase activity andsubstantially reduced RNase H activity made using recombinant DNAtechniques wherein the RT is modified using point mutations. Moreparticularly, the present invention includes RTs with one or more pointmutations in the nucleotide selection domain, RTs with one or more pointmutations in the processivity domain, and hyperactive RTs that includeRTs with mutations in both the nucleotide selection and the processivitydomains. The RTs disclosed herein may be expressed in host cells using,e.g., recombinant plasmids constructed as described herein that providereverse transcriptase for use in recombinant DNA technology tosynthesize cDNA from mRNA without the unwanted effects of RNase Hactivity, which can excessively degrade the mRNA template duringfirst-strand synthesis.

As used herein, the terms “hyperactive Reverse Transcriptase,”“hyperactive RT” and the like are used to describe a hyperactive RTpurified to near homogeneity and having the ability to enable greaterthan 20% more amplified RNA that can be generated by the polymeraseactivity of a wild-type RT DNA polsymerase domain from an input of 100ng total RNA in an RNA amplification reaction that includes a 4 hr invitro transcription reaction. For example, an isolated hyperactiveReverse Transcriptase that includes one or more point mutations in the“processivity domain” and one or more point mutations in the “nucleotideselection domain” is able to generate a yield of greater than 5micrograms of aRNA in a single amplification reaction from 100 ng oftotal RNA, e.g., in a single round RNA amplification of 100 ng HeLa S-3Total RNA or Rat Thymus Total RNA against wild-type enzyme MMLV-RT, AMVRT or any other RT as determined by, e.g., UV absorbance at 260 nm orother equivalent methods known to the skilled artisan. As will beapparent to those of skill in the art, the “hyperactivity” of theenzymes of the present invention may be as quantitatively distinct dueto, e.g., assay conditions, temperatures, times, salts, source of RNA,quality of RNA, activity read-out and the like.

The term “processivity domain” is used to describe the region of the RTthat is responsible for maintenance of the template integrity in astandard RT reaction. As defined herein, the processivity domainincludes amino acids 497-671. One indication of processivity is theaverage length of the cDNA that can be synthesized from a long mRNAtarget. The present invention is distinct from the domains identifiedby, e.g., Gerard, et al., U.S. Pat. No. 5,668,005 and patents relatedthereto, which functionally identified the region spanning MMLV RT aminoacids 503 through 611 as critical for RNase H activity. In contrast tothe region identified by Gerard, et al., the present inventionidentifies locations and mutations outside of this previouslycharacterized region as also affect RNase H activity and, importantly,enzyme processivity. Indeed, the mutant RT enzymes described hereincatalyze yields of amplified RNA that are superior to other,commercially available enzymes mutated in the RNase H domain, such asSuperScript II. As a result, the inventors describe novel mutations thatenable a large and unexpected improvement in the yield of amplified RNAin Eberwine-like RNA amplification protocols. The hyperactive RT withmutations in the processivity domain of the RTs may also include one ormore point mutations in other domains. The present inventors haveidentified one series of mutants that can affect the sensitivity of theRT for distinguishing or having a preference for ribonucleotides and/ordeoxyribonucleotides during DNA synthesis, which are described herein asthe “nucleotide selection domain.” As used herein the phrase “nucleotideselection domain” or “NSD” includes but is not limited to, mutations inthe following amino acids in MMLV-RT: D153, A154, F155, F156, C157, orL158 and the equivalent mutations in other RTs. The equivalent mutationin the other RTs may be localized based on the crystal structure of MMLVRT, which reveals a secondary structure motif that encompasses a 3₁₀helix around the nucleotide selection domain namely, amino acids 153 to158 of the MMLV-RT.

Described herein are point mutations that alone or in combinationsignificantly enhance the yield of amplified nucleic acids used usefulfor, e.g., the amplification of isolated RNA for use in nucleic acidmicroarrays. Another method to detect the activity of the hyperactiveRTs of the present invention is the length of the cDNAs, wherein thehyperactive RTs are able to copy an mRNA to a product length greaterthan 9, 11, 15 or even 20 kilobases.

As used herein, the term “substantially reduced RNase H activity” isused to describe an RT purified to near homogeneity and having an RNaseH activity of between about 0.01%, 1, 3, 4, 6, 9, 10, 15, 20, 25 and 50%of the RNase H activity of a wild-type RT RNase H domain. Describedherein are point mutations that alone or in combination reduce the levelof degradation of the RNA template used in an RT reaction, that is,without significant degradation of the mRNA template during first-strandsynthesis, but that maintain “processive” activity. The term“processivity” as used herein is used to describe the ability of the RTto elongate its nucleic acid product to produce a longer product. Thisprocessivity domain includes, but is not limited to, one or more of thefollowing mutations corresponding to the amino acids in MMLV-RT: H638G,Y586A, D653N, D524N, D524E and E562D. The double mutants of the presentinvention also include mutations to the processivity domaincorresponding to the amino acids in MMLV-RT: H638G, Y586A, D653N, D524N,D524E and E562D and one or more mutations to the nucleotide selectiondomain that include one or more of the following mutations in thefollowing amino acids in MMLV-RT: D153, A154, F155, F156, C157, or L158.

The term “degenerate variants” as used herein describes havingvariations in the DNA or amino acid sequence that vary the amino acidsat the processivity domain and the nucleotide selection domain such thatthe activities described herein are maintained. The term“codon-optimized” sequence is used to describe a hyperactive RT in whichat least a portion of the sequence has been modified by directedsequence modification, for example, changes to the sequence in one ormore underlying sequences that may or may not affect the amino acidsequence but that are use to, e.g., improve the expression of theprotein by using codons that are more commonly used in a particular hostorganism. By the term “recombinant,” “isolated,” “cloned” hyperactive RTor grammatical equivalents herein is meant a polypeptide having amodified nucleic or amino acid sequence of a mature RT (for example,from about 85 to 100% identical) as described herein, as well as aminoacid sequence variants that are enzymatically active RNA directed, DNApolymerases with a catalytic profile that is distinct from that of wildtype RT, e.g., AMV RT, MMLV RT and the like as defined hereinabove. Inaddition, sequences may be the combination of sequences from differentorganisms for the same or closely related sequences to, e.g., modify thefunctionality of the final protein by directed modifications or even topermit specific recombinant modification or manipulation by the user.

As defined herein, a “wild type” sequence, whether found in a coding,non-coding or interface sequence is an allelic form of sequence thatperforms the natural or normal function for that sequence. Therefore, asused herein a wild type sequence includes multiple allelic forms of acognate sequence, for example, multiple alleles of a wild type sequencemay encode silent or conservative changes to the protein sequence that acoding sequence encodes. A “mutant” sequence is defined herein as one inwhich at least a portion of the functionality of the sequence has beenlost, for example, changes to the sequence in a promoter or enhancerregion will affect at least partially the expression of a codingsequence in an organism. A “mutation” in a sequence as used herein isany change in a nucleic acid sequence that may arise such as from adeletion, addition, substitution, or rearrangement. The mutation mayalso affect one or more steps that the sequence is involved in. Forexample, a change in a DNA sequence may lead to the synthesis of analtered protein, one that is inactive, or to an inability to produce theprotein. A “mutation frequency” as used herein is the frequency or ratewith which a particular mutation appears in a particular dataset.Mutation frequency may also be the frequency at which any mutationappears in the whole dataset.

A sample is any mixture of macromolecules obtained from a solution, acell culture, a supernatant, an animal, an environmental sample, a foodsample or even a patient. This also includes separated fractions of allof the preceding. Examples of samples include, but are not limited to,blood, plasma, urine, semen, saliva, lymph fluid, meningeal fluid,amniotic fluid, glandular fluid, and cerebrospinal fluid. “Sample” alsoincludes solutions or mixtures containing homogenized solid material,such as feces, cells, tissues, and biopsy samples. Samples hereininclude one or more that are obtained at any point in time, includingdiagnosis, prognosis, and periodic monitoring.

The terms “a sequence essentially as set forth in SEQ ID NO.: (#)”, “asequence similar to”, “nucleotide sequence” and similar terms, withrespect to nucleotides, refers to sequences that substantiallycorrespond to any portion of the sequence identified herein as SEQ IDNO.: 1 or the point mutants and combination of point mutants of RTsdescribed herein and the functional counterparts in related RTs. Theseterms refer to synthetic as well as naturally-derived molecules andincludes sequences that possess biologically, immunologically,experimentally, or otherwise functionally equivalent activity, forinstance with respect to hybridization by nucleic acid segments, or theability to encode all or portions of an RT having DNA polymerase and/orsubstantially reduced RNase H activity. Naturally, these terms are meantto include information in such a sequence as specified by its linearorder.

The term “homology” refers to the extent to which two nucleic acids arecomplementary. There may be partial or complete homology. A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The degree or extent of hybridization may be examined usinga hybridization or other assay (such as a competitive PCR assay) and ismeant, as will be known to those of skill in the art, to includespecific interaction even at low stringency.

The inhibition of hybridization of the completely complementary sequenceto the target sequence may also be examined using a hybridization assayinvolving a solid support (e.g., Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Lowstringency conditions may be used to identify the binding of twosequences to one another while still being specific (i.e., selective).The absence of non-specific binding may be tested by the use of a secondtarget that lacks even a partial degree of complementarity (e.g., lessthan about 30% identity). In the absence of non-specific binding, theprobe will not hybridize to the second non-complementary target and theoriginal interaction will be found to be selective. Low stringencyconditions are generally conditions equivalent to binding orhybridization at 42 degrees Centigrade in a solution consisting of5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH₂PO₄—H₂O and 1.85 g/l EDTA, pH 7.4),0.1% SDS, 5×Denhardt's reagent (50×Denhardt's contains per 500 ml: 5 gFicoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma) and 100micrograms/ml denatured salmon sperm DNA); followed by washing in asolution comprising 5×SSPE, 0.1% SDS at 42 degrees Centigrade when aprobe of about 500 nucleotides in length is employed. The art knows thatnumerous equivalent conditions may be employed to achieve low stringencyconditions. Factors that affect the level of stringency include: thelength and nature (DNA, RNA, base composition) of the probe and natureof the target (DNA, RNA, base composition, present in solution orimmobilized, etc.) and the concentration of the salts and othercomponents (e.g., formamide, dextran sulfate, polyethylene glycol).Likewise, the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, inclusion of formamide, etc.).

The term “gene” is used to refer to a functional protein, polypeptide orpeptide-encoding unit. As will be understood by those in the art, thisfunctional term includes genomic sequences, cDNA sequences, or fragmentsor combinations thereof, as well as gene products, including those thatmay have been altered by the hand of man. Purified genes, nucleic acids,protein and the like are used to refer to these entities when identifiedand separated from at least one contaminating nucleic acid or proteinwith which it is ordinarily associated. As used herein the terms“protein”, “polypeptide” or “peptide” refer to compounds comprisingamino acids joined via peptide bonds and are used interchangeably.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Thevector may be further defined as one designed to propagate specificsequences, or as an expression vector that includes a promoteroperatively linked to the specific sequence, or one designed to causesuch a promoter to be introduced. The vector may exist in a stateindependent of the host cell chromosome, or may be integrated into thehost cell chromosome

The term “host cell” refers to cells that have been engineered tocontain nucleic acid segments or altered segments, whether archeal,prokaryotic, or eukaryotic. Thus, engineered, or recombinant cells, aredistinguishable from naturally occurring cells that do not contain genesintroduced recombinantly through the hand of man.

The term “altered”, or “alterations” or “modified” with reference tonucleic acid or polypeptide sequences is meant to include changes suchas gross or point: insertions, deletions, substitutions, fusions withrelated or unrelated sequences, such as might occur by the hand of man,or those that may occur naturally such as polymorphisms, alleles andother structural types. Alterations encompass genomic DNA and RNAsequences that may differ with respect to their hybridization propertiesusing a given hybridization probe. Alterations of polynucleotidesequences for a hyperactive reverse transcriptase, or fragments thereof,include those that increase, decrease, or have no effect onfunctionality. Alterations of polypeptides refer to those that have beenchanged by recombinant DNA engineering, chemical, or biochemicalmodifications, such as amino acid derivatives or conjugates, orpost-translational modifications.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it effects the transcription of the sequence; ora ribosome binding site is operably linked to e coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in same reading frame.Enhancers do not have to be contiguous Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

As used herein, the expressions “cell” and “cell culture” are usedinterchangeably end all such designations include progeny. Thus, thewords “transformants” and “transformed cells” include the primarysubject cell and cultures derived therefrom without regard for thenumber of transfers. It is also understood that all progeny may not beprecisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Different designations are will be clear from the contextuallyclear.

“Plasmids” are designated by, e.g., a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare commercially available, are publicly available on an unrestrictedbasis, or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

“Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” The term“vector” as used herein also includes expression vectors in reference toa recombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably-linked coding sequence in a particular host organism. Nucleicacid sequences necessary for expression in prokaryotes usually include apromoter, an operator (optional), and a ribosome binding site, oftenalong with other sequences. The choice of a suitable vector depends on anumber of considerations known to one of ordinary skill in the art, suchas the size of the fragment, nature of the host, number and position ofrestriction sites desired, and the selection of marker and markersdesired for selection. Expression of the RT genes may also be placedunder control of other regulatory sequences homologous or heterologousto the host organism in its untransformed state as will be known to theskilled artisan. The selection of the host cell for transformation mayinfluence the decision of which vector and/or regulatory sequences areprovided along with the RT construct. Eukaryotic cells are known toutilize promoters, enhancers, and termination and polyadenylationsignals.

As used herein, the term “amplify”, when used in reference to nucleicacids refers to the production of a large number of copies of a nucleicacid sequence by any method known in the art. Amplification is a specialcase of nucleic acid replication involving template specificity.Template specificity is frequently described in terms of “target”specificity. Target sequences are “targets” in the sense that they aresought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primermay be single stranded for maximum efficiency in amplification but mayalternatively be double stranded. If double stranded, the primer isfirst treated to separate its strands before being used to prepareextension products. The primer must be sufficiently long to prime thesynthesis of extension products in the presence of the inducing agent.The exact lengths of the primers will depend on many factors, includingtemperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g. ELISA, as well as enzyme-based histochemicalassays), fluorescent, radioactive, and luminescent systems. It is notintended that the present invention be limited to any particulardetection system or label.

As used herein, the term “target” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted oat from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and4,965,188, hereby incorporated by reference, which describe a method forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”. With PCR, it is possible to amplify a single copy ofa specific target sequence in genomic DNA to a level detectable byseveral different methodologies (e.g., hybridization with a labeledprobe; incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of ³²P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide sequence can be amplifiedwith the appropriate set of primer molecules. In particular theamplified segments created by the PCR process itself are, themselves,efficient templates for subsequent PCR amplifications.

The reverse transcriptase gene (or the genetic information containedtherein) can be obtained from a number of different sources, e.g.,Moloney Murine leukemia virus (M-MLV); human T-cell leukemia virus typeI (HTLV-I); bovine leukemia virus (BLV); Rous Sarcoma Virus (RSV); humanimmunodeficiency virus (HIV); yeast, including Saccharomyces,Neurospora, Drosophila; primates; and rodents. See, e.g., Weiss et al.,U.S. Pat. No. 4,663,290 (1987); Gerard, G. R., DNA 5:271-279 (1986);Kotewicz, M. L., et al., Gene 25:249-258 (1985); Tanese, N., et al.,Proc. Natl. Acad. Sci. (USA) 82:4944-4948 (1985); Roth, M. J., et al.,J. Biol. Chem. 260:9326-9335 (1985); Michel, F., et al., Nature316:641-643 (1985); Akins, R. A., et al., Cell 47:505-516 (1986), EMBOJ. 4:1267-1275 (1985); and Fawcett, D. F., Cell 47:1007-1015 (1986). Forinstance, the gene may be obtained from public sources, e.g., ATCC, ormay even be purified from eukaryotic cells infected with a retrovirus,or from a plasmid that includes a portion the retrovirus genome thatincludes the RT.

The mutation(s) for producing a hyperactive polymerase domain asdescribed herein may be obtained by point mutation(s) in theprocessivity domain as described and disclosed herein. Likewise, RTgenes having DNA polymerase activity and substantially reduced RNase Hactivity may be obtained by point mutation(s) of the nucleotideselection domain. The plasmid thus obtained may then be used totransform hosts which may then be screened for hyperactive RT activity.RT RNase H activity may also be assayed by template solubilization ascompared to, e.g., a wild-type AMV, MMLV or other RT.

The invention also includes fusion proteins that include the hyperactivereverse transcriptase of the invention with, e.g., a carrier protein orother anchor domain that permits isolation and purification. It is alsopossible to prepare fusion proteins of the hyperactive reversetranscriptase that are substituted at the amino or carboxy termini withpolypeptides which stabilize or change the solubility of the reversetranscriptase. Amino-terminal and carboxy-terminal gene fusion proteindomains are well known in the art.

The transformed hosts of the invention may be cultured under, e.g.,protein producing conditions according to any of the methods that areknown to those skilled in the art or protein production, purification,isolation and characterization. Of particular use may be host cells thathave reduced endogenous RNase activity, e.g., as taught in U.S. Pat. No.5,998,195, relevant host cells, constructs, vectors and methodsincorporated herein by reference. The hyperactive RT of the presentinvention may be isolated according to conventional methods known tothose skilled in the art. For example, after protein expression the hostcells may be collected by centrifugation, washed with suitable buffers,lysed, and the reverse transcriptase isolated by column chromatography,for example, on DEAE-cellulose, phosphocellulose or other standardisolation and identification techniques using, for example,polyribocytidylic acid-agarose, or hydroxylapatite or by electrophoresisor immunoprecipitation.

The hyperactive RT of the present invention may be used with any assayand included in any kit that calls for an RT, e.g., it may be used toprepare cDNA from RNA by hybridizing a primer, e.g., an oligo(dT)primer, or other complementary primers with the mRNA. The hyperactive RTof the present invention is particularly useful for the synthesis offull-length and/or extra-long length, complete cDNA by adding thehyperactive RT and all four deoxynucleoside triphosphates underconditions that permit elongation. Using the hyperactive RT produced bythe present invention allows for the preparation of cDNA from mRNA withreduced degradation of the mRNA, which results in cDNA synthesis ofmessages exceeding 9, 12 or even 15 kilobases.

The hyperactive RT of the present invention is suited for incorporationinto a kit for the preparation of cDNA from RNA, for aRNA synthesis andfor amplification of mRNA for microarray analysis. Such a kit willgenerally include one or more containers, such as vials, tubes, and thelike, and the kit will containers that have alone or in combination oneor more of the separate elements of the method used, e.g., to preparecDNA from RNA or for amplification of the RNA. For example, the kit mayinclude one vial that has the hyperactive RT in solution. Separatecontainers may include suitable buffers, substrates for DNA synthesissuch as the deoxynucleotides, oligo(dT) primer, and even a control RNAfor use as a standard.

The reverse transcriptase may be present in the solution at aconcentration of 5, 10, 100, 200, 400 or more units/ml. The reversetranscriptase may also be lyophized in a plate well, and its activityreconstituted upon hydration of the lyophilized enzyme. Deoxynucleotidesmay be present either in lyophilized form, as part of a buffer or insolution at a concentration of, e.g., about 0.5 mM to about 2 mM each. Asuitable buffer, present at 2, 5, 10, 50 and/or 100 times the finalconcentration of use may be, e.g., 250 mM Tris-HCl (pH 7.5 to 8.3), 375mM KCl, 15 mM MgCl₂, and 50 mM dithiothreitol. An oligo (dT) may bepresent at a concentration of 5 ug/ml to 20 ug/ml. Control RNA, such as2.3, 9.0 or greater kilobase control RNA, may be present at aconcentration of 10 ug/ml to 20 ug/ml.

Reverse transcriptase-polymerase chain reaction (RT-PCR) and/orsimultaneous DNA cleavage and reverse transcription may be conductedusing the hyperactive RT. The hyperactive RT of the present inventionmay used in conjunction with standard RT-PCR techniques. RT-PCR is acommon molecular biology procedure that typically requires DNA-free RNA.DNase I digestion of contaminating DNA is the method of choice foreradicating DNA in RNA preparations destined for reverse transcriptionand PCR.

Example 1

The present inventors recognized that current RT mutants fail to providethe best combination of amplification, processivity, fidelity and easeof use. As such, the inventors focused on the creation of plasmidsexpressing H638G MMLV RT, Y586A MMLV RT, D653N MMLV RT, D524N MMLV RT,D524E MMLV RT, and E562D MMLV RT. Efforts to develop an improved RT forRNA amplification began with strategies for modulating RT-associatedRNase H activity. MMLV and AMV-related RTs with no RNase H activity areknown to synthesize longer cDNA products than their RNase H⁺counterparts. However, a complete loss of RNase H activity may haveuntoward effects in some applications (for an example, see Biotechniques2002 June; 32(6): 1224-5). In fact, RNase H treatment of first strandcDNA is an obligate step in the aRNA synthesis procedure. Thus, anappropriately balanced ratio of RNase H to polymerase activity waspotentially desirable.

As MMLV RT is modified easily by molecular techniques, this enzyme wasthe target for improvement efforts. FIG. 1 shows the relative domainstructure of MMLV RT with the relevant point mutations of the presentinvention marked in relation to the domains. The present inventorsidentified a number of amino acid residues within the DNA polymerasedomain and carboxy from the RNase H domain of MMLV RT as potentialtargets for site-directed mutagenesis. This domain is composed of aportion of the carboxy-terminus of the RT. For example, one group hasidentified amino acid residues 503-611 as critical for RNase H activityby gross deletion. Although the three dimensional structure of MMLV RTRNase H domain has not been solved, the corresponding structure of E.coli RNase H1 is known (Science. 1990 Sep. 21; 249(4975):1398-405).However, E. coli RNase H1 shares only 30% identity with the MMLV RNase H(Proc Natl Acad Sci USA. 1986 October; 83(20): 7648-52), which includeessential metal binding and active site residues. Several “support”residues not directly involved in catalysis were also identical in thetwo enzymes. Several mutants of E. coli RNase H1 have been identifiedthat exhibit reduced RNase H activity. The data provided herein supportthe choice of 9-10 mutants that were found to enhance the ability of theRT to maintain template interactions, significantly reduce the RNase Hactivity of MMLV RT to a level in the 1-50% range as compared to thewild-type MMLV RT, but not having the deleterious effects of deletionmutants or mutants having no RNase H activity. The results disclosedherein demonstrate that reduced activity (but not eliminated or no RNaseH activity), is desirable for the aRNA synthesis application and thecreation of a hyperactive RT.

The present inventors have developed a series of point mutants, e.g.,H638G MMLV RT, Y586A MMLV RT, D653N MMLV RT, D524N MMLV RT, D524E MMLVRT, and E562D MMLV RT using pSE380 containing the MMLV RT gene(pSE380-MMLV RT) and the mutagenic primers given in Table 1. The nucleicacid sequence for one such mutant is shown in FIG. 2, with the aminoacid sequence described in FIG. 3. Amplification of the mutant sequenceswas accomplished via PCR using the Quick Change mutagenesis kit(Stratagene). The resulting PCR product was transformed and plated ontosolid media containing ampicillin Plasmid DNA from selected clones wasprepared with the QIAprep Spin Miniprep Kit. In the case of Y586A MMLVRT and H638G MMLV RT, the presence of the correct mutation was diagnosedafter restriction digest with Sma I. Clones containing D653N, D524N MMLVRT, D524E MMLV RT, and E562D MMLV RT were screened by sequencing. Ineach case, sequencing across the MMLV gene confirmed the desiredmutations.

TABLE 1  Mutagenic Primers Used to Create H638G MMLV RT, Y586A MMLV RT, and D653N MMLV RT.  H638G-FCTTAGCATAATCCATTGTCCCGGGGGTCAAAAGGGACACAGCGC  (SEQ ID NO.: 3); H638G-RGCGCTGTGTCCCTTTTGACCCCCGGGACAATGGATTATGCTAAG  (SEQ ID NO.: 4); Y586A-FGAAGCTAAATGTTTATACTGATTCCCGGGCTGCTTTTGCTACTGCCC (SEQ ID NO.: 5); Y586A-RGGGCAGTAGCAAAAGCAGCCCGGGAATCAGTATAAACATTTAGCTTC (SEQ ID NO.: 6); D653N-FGGCAACCGGATGGCTAACCAAGCGGCCCGAAAG (SEQ ID NO.: 7); D653N-RCTTTCGGGCCGCTTGGTTAGCCATCCGGTTGCC (SEQ ID NO.:  8); D524E-FCACACCTGGTACACGGAAGGAAGCAGTCTCTTAC (SEQ ID  NO.: 9); D524E-RGTAAGAGACTGCTTCCTTCCGTGTACCAGGTGTG (SEQ ID  NO.: 10); D524N-FCACACCTGGTACACGAATGGAAGCAGTCTCTTAC (SEQ ID  NO.: 11); D524N-RGTAAGAGACTGCTTCCATTCGTGTACCAGGTGTG (SEQ ID  NO.: 12); E562D-FCGCTCAGCGGGCTGATCTGATAGCACTCACCC (SEQ ID NO.:  13); and E562D-RGGGTGAGTGCTATCAGATCAGCCCGCTGAGCG (SEQ ID NO.:  14). “F” and “R” refer to“forward” and “reverse” primers, respectively.

Example 2

Creation of plasmids expressing F155Y MMLV RT, R301L MMLV RT, and F309AMMLV RT. Clones F155Y MMLV RT, R301L MMLV RT, and F309A MMLV RT werecreated using pSE380 containing the MMLV RT gene (pSE380-MMLV RT) andthe mutagenic primers given in Table 2. Amplification of the mutantsequences was accomplished via PCR using the Quick Change mutagenesiskit (Stratagene). The resulting PCR product was transformed and platedonto solid media containing ampicillin Plasmid DNA from selected cloneswas prepared with the QIAprep Spin Miniprep Kit. For each mutant,sequencing across the MMLV gene confirmed the desired mutations.

TABLE 2 Mutagenic Primers Used to Create F155Y MMLV RT,R301L MMLV RT, and F309A MMLV RT.  F155Y-FGATTTAAAGGATGCCTATTTCTGCCTGAGACTC (SEQ ID NO.:  15); F155Y-RGAGTCTCAGGCAGAAATAGGCATCCTTTAAATC (SEQ ID NO.:  16); R301L-FGACCCCTCGACAACTACTGGAGTTCCTAGGGACGGC (SEQ ID  NO.: 17); R301L-RGCCGTCCCTAGGAACTCCAGTAGTTGTCGAGGGGTC (SEQ ID  NO.: 18); F309A-FTCCTAGGGACGGCAGGCGCCTGTCGCCTCTGGATCCCTG (SEQ  ID NO.: 19); and F309A-RCAGGGATCCAGAGGCGACAGGCGCCTGCCGTCCCTAGGA (SEQ  ID NO.: 20). “F” and “R”refer to “forward” and “reverse” primers, respectively.

Example 3

Creation of Multiple Mutated MMLV RT Enzymes: Combined Polymerase andRNase H Mutations. To create combined MMLV RT mutants, plasmidscontaining the single mutations were used as templates for asecond-round and/or third-round mutagenesis reaction. For example, tocreate the F155Y;H638G MMLV RT mutant, the following changes to the RTgene were made, beginning with wild-type MMLV RT (Accession numberJ02255):

1) Wild-type MMLV RT gene→Change F155 to Y155

2) F155Y MMLV RT→Change H638 to G638 Example 4

Expression and Purification of MMLV RT Mutants. Plasmids carrying eachmutated MMLV RT gene were transformed into XL-1 Blue E. coli cells.Single colonies were picked, and cultured overnight in LB mediacontaining Ampicillin The next day, 5 ml of the culture was used toinoculate 0.5-4 L of LB-Amp. Cells were grown to A600-0.4 at ˜29° C.with 250 rpm shaking, and then induced with IPTG. After 12-16 hr growthat ˜29° C., cell pellets were harvested for purification.

The MMLV mutants may be isolated and purified using a multitude oftechniques known by the skilled artisan depending, e.g., in the level ofpurity desired and the expected uses of the MMLV. Examples of methods ofpurification include, e.g., crude filtration, column purification,epitope tagging, isolation by specific or non-specific binding toresins, selective secretion and the like. In one example, purificationof the MMLV RT mutants was accomplished by resuspending frozen cellsfrom cultures in a buffered, ionically controlled solution, e.g., abuffer containing 20 mM KPi pH 7.0, 500 mM NaCl and a proteaseinhibitor, e.g., 1 mM PMSF. The contents of the resuspended cells arethen extracted using standard methods, e.g., French press, shearing oreven lysozyme digestion 4 C for 30 min, followed by sonication or otherforms of mechanical stress. The cell debry may then be cleared bycentrifugation or filtration. Examples of well-known techniques forcellular content release such as cellular permeabilization aresummarized in, e.g., U.S. Pat. No. 6,630,333, relevant portionsincorporated herein by reference.

Following the release of the hyperactive reverse transcriptase enzymesof the present invention, whether alone or as fusion proteins, a varietyof protein purification techniques may be followed that are well-knownto one of ordinary skill in the art. Suitable techniques forpurification include, but are not limited, e.g., ammonium sulfate and/orethanol precipitation, acid extraction, preparative gel electrophoresis,immunoadsorption, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, immunoaffinity chromatography, size exclusionchromatography, liquid chromatography (LC), high performance LC(HPLC),fast performance LC (FPLC), hydroxylapatite chromatography, lectinchromatography, binding to Glutathione-S-Transferase-resin (GST-resin),Maltose-resin and immobilized metal affinity chromatography (IMAC).Generally, the hyperactive reverse transcriptase will be purified by acombination of liquid chromatographic techniques including ion exchange,affinity and size exclusion. When using a tagged fusion protein, thehyperactive reverse transcriptase may be released by, e.g., proteasedigestion of a linker, addition of a competitor (GST or Maltose),addition of a chelating agent (IMAC) and the like, depending on thesystem used.

Alternative chromatographic solid supports, mobile phases and associatedmethods may be equivalently used and will be well-known to one ofordinary skill of protein isolation and purification. The invention thusprovides for substantially isolated and purified hyperactive reversetranscriptase. Substantially pure a used herein refers to a preparationor sample which is substantially free of contaminating components,proteins, etc., which may adversely affect the activity or performanceof the hyperactive reverse transcriptase in the use of the enzyme suchas in amplification or synthesis. If the hyperactive reversetranscriptase if produced as a fusion protein, the skilled artisan mayselect any of a number of well-known fusion partners, e.g., GST, MBP,FLAG, myc, H is or other tagging methodologies and/or techniques.Depending on the fusion partner and/or tag, the supernatant is loaded onthe appropriate column under specific ionic and buffering conditions andthe hyperactive RT protein allowed to bind, followed by isolation of thehyperactive RT of, or from, the fusion peptide/protein carrier. Forexample, in some cases the hyperactive RT remains on the column and thefusion protein/peptide carrier is in the flow-through or vice versa, aswill be known to the skilled artisan. In some embodiment, thehyperactive RT may still be used while on or about the resin, that is,as a hyperactive RT resin.

Example 5

Measurement of RT-associated RNase H Activities. Characterization of theRNase H activity of the MMLV RT mutants was shown using an assay thatreports cleavage of RNA from an RNA:DNA hybrid. Briefly, a hybridsubstrate was created by annealing a 1.5 kb RNA with an internal 20-baseDNA oligonucleotide to a sequence that is 500 bp from the end of theRNA. Scission of the RNA results in two fragments, 1.0 kb and 0.5 kb,which are resolved and quantified on an RNA LabChip. The assay (5 ul)contains 2 uM DNA, 100 ng/ul RNA, in 1 xRT buffer. RNase H activity ofeach mutant MMLV RT was compared by monitoring the fraction of cleavageas a function of time.

Table 4 shows the RNase H activity of MMLV RT Mutants. RNase H activityis expressed as the number of polymerase unit enzyme equivalents thatachieved 50% cleavage of the RNA:DNA hybrid substrate. In most cases,cleavage was quantified at ˜20-35% cleavage (when the assay was trulylinear with respect to time and input protein) and extrapolated to 50%to provide a convenient mathematical reference point. ND=Not detected.

TABLE 4 # Pol Units (U/ug) to Achieve % of Specific RNase H Source 50%Cleavage wt-MMLV Activity* E. coli RNase H 0.005 (Ambion-Cloned) AMV(Ambion) 2.0 35 MMLV-RT (Ambion) 0.7 100  200 Powerscript (Clontech) 100ND <<<<1%**   U/rxn Superscript II (Invitrogen) ND <<<<1%**   400 U/rxnD524E MMLV RT 5 U/rxn ND ~1% 121 D524N MMLV RT 5 U/rxn ND <1% 147 E562DMMLV RT 5 U/rxn 23 ~3% 135 D653N MMLV RT 4.5 16 192 H638G MMLV RT 5.2 13236 F155Y; H638G-MMLV RT 7.0 10 240 F155Y MMLV RT 0.8 85 240 *Units/ugprotein. **No detectable activity.

Example 6

Analysis of cDNA Product Synthesis by MMLV RT Mutants. An importantproperty of RT enzymes is that they are able to synthesis cDNA productsfrom mRNA that faithfully maintains the original information content ofthe transcript. In other words, these enzymes should exhibit a highapparent processivity. Several of the MMLV RT single mutants (D524E,D524N, E562D, H638G, D653N, R301L and F309A) were compared with wildtype MMLV-RT (Ambion), wt-MMLV-RT-His and SuperScriptII and III fortheir ability to make long cDNA products using Ambion's Millenium MarkerRNA templates (ranging in size from 0.5 kb to 9.0 kb). The results areshown in FIG. 4, which is a gel that shows a comparison of cDNA productlengths by various mutant MMLV RT enzymes using RNA templates from 0.5to 9.0 kb in Size.

Briefly, 20 ul reactions included 500 ng of millenium marker and 10pmole of oligo dT (annealing at 70° C. for 5 min and cool to 42° C.),250 μM dGTP, dCTP, dTTP, 25 μM dATP, 0.5 μl α-³²P dATP (3000 μCi/mM), 8units of RIP and the indicated amount (10 or 100 U) of MMLV-RT enzyme inAmbion's RetroScript buffer. The reaction was incubated at 42° C. for 1hr and stopped by heating at 95 C for 5 min. A total of 5 μl of samplewas mixed with an equal volume of glyoxal loading dye, heated at 50° C.for 30 min, and resolved on 1% Agarose-glyoxal gel. The products weretransferred to a nylon membrane prior to exposure to film. Lanes 1 and11: Ambion MMLV-RT; lanes 2 and 12 MMLV-RT-His; lanes 3 and 13 D524EMMLV RT; lanes 4 and 14 D524N MMLV RT; lane 5 and 15: E562D MMLV RT;lanes 6 and 16: H638G MMLV RT; lanes 7 and 17: D653N MMLV RT; lanes 8and 18: R301L MMLV RT; lanes 9 and 19: F309A MMLV RT; lanes 10 and 20:SuperScriptll. Lane 18* has only 20 U D653N MMLV RT instead of 100 Uowing the lower stock concentration of this mutant.

Other MMLV RT mutants were characterized in similar assays. For example,cDNA products by F155Y MMLV RT, H638G MMLV RT, D524E MMLV RT, D524N MMLVRT, E562D MMLV RT, D653N MMLV RT and the double mutant F155Y;H638G MMLVRT were assayed in a reaction that uses a higher concentration of evenlybalanced dNTP's and a single, 9.0 kb RNA template. FIG. 5 is a gel thatshows a comparison of cDNA product lengths by various mutant MMLV RTenzymes using a 9.0 kb RNA template. For these reactions,template-primer was incubated for 4 min at 70° C., and then added to thereaction mastermix at 42° C. Reactions were initiated by adding 50 U ofenzyme and incubating for 30 min at 42° C. Residual, unhybridized RNAtemplate was removed by treating all reactions with 500 pg/ul bovineRNase A treat in combination with a 1:10,000 dilution of SYBR Gold (tostain the cDNA products). Samples were treated with RNase for 30 min at37° C. A total of 1 μl of 10×DNA loading dye was added, and one-half ofthe reaction mixture loaded onto 0.7% agarose gel. The cDNA productswere resolved after electrophoresis for 40 min at 90V.

The top half of the gel in FIG. 5 shows the cDNA products with these RTenzymes in a buffer containing 200 ng 9 kb RNA template, 5 uM oligo dTprimer, 50 mM Tris pH 8.3, 75 mM KCl, 5 mM DTT, 0.5 mM of each dNTP, 10U RIP, and 50 U RT enzyme in a 20 ul reaction volume. The bottom half ofthe gel shows cDNA products from identical reactions, except that 3 mMMnCl₂ was used instead of 3 mM MgCl₂. This change in the divalent ionchanges the product profile significantly, since Mn²⁺ is known todramatically enhance RNase H activity. As a result, Mn²⁺ causes the cDNAproducts to be shorter in proportion to the extent of RNase H activityextant in each RT. As a result, those mutants with the greatest amountof RNase H activity make the shortest cDNA products, and those enzymesthat have even 10-15% RNase H activity are readily distinguished fromenzymes with <1% RNase H activity.

It is significant to note that although the F155Y;H638G MMLV RT mutantfails to demonstrate an increase in this cDNA length assay with thislimited length template (9 kb), it was found to outperform all otherMMLV RT mutant enzymes in RNA amplification.

Example 7

RNA Amplification Properties of MMLV RT Mutants. The RNA amplificationreagents used were from Ambion's MessageAmp kit (Ambion, Inc., Austin,Tex., Cat#1750). The reactions were performed according to theinstruction manual with 100 units of RT and indicated amount oftemplate. Briefly, the MessageAmp (Ambion, Inc., Austin, Tex.) procedureis based on antisense RNA (aRNA) amplification and involves a series ofenzymatic reactions resulting in linear amplification of exceedinglysmall amounts of RNA for use in array analysis. Unlike exponential RNAamplification methods, such as NASBA and RT-PCR, aRNA amplificationmaintains representation of the starting mRNA population.

The procedure begins with total or poly(A) RNA that is reversetranscribed using a primer containing both oligo(dT) and a T7 RNApolymerase promoter sequence. After first-strand synthesis, the reactionis treated with RNase H to cleave the mRNA into small fragments. Thesesmall RNA fragments serve as primers during a second-strand synthesisreaction that produces a double-stranded cDNA template fortranscription. Contaminating rRNA, mRNA fragments and primers areremoved and the cDNA template is then used in a large scale in vitrotranscription reaction to produce linearly amplified aRNA. The aRNA canbe labeled with biotin rNTPS or amino allyl-UTP during transcription.Alternatively, unlabeled aRNA can be used as a template for a reversetranscription with CyDye™-labeled dNTPs to generate labeled cDNA. TheRETROscript™ Kit (Ambion, Inc.) may be used for this purpose. Forincreased yields, the aRNA can also be used as template for cDNAsynthesis followed by a second round of amplification using MessageAmp.

FIG. 6 is a graph of a single round RNA amplification with: (100 ng RatThymus Total RNA, comparing different MMLV RT mutants versus wild-type.The aRNA yields were determined by UV absorbance at 260 nm. Samples wereperformed in duplicate compares the aRNA yields from each of the MMLV RTmutants. Significantly, the double mutant F155Y;H638G MMLV RT producedabout 3 to 5 times more aRNA than several other enzymes tested, such asMMLV RT (Ambion), AMV RT (Ambion), and Superscript II (Invitrogen). In aseparate study using 100 ng of human HeLa-S3 cell total RNA, theF155Y;H638G MMLV RT produced 1.5 to 2.2 times more aRNA compared toother RTs, including AMV RT and SSII, after one round of amplification.FIG. 7 is a graph of a single round RNA amplification with 100 ng HeLaS-3 Total RNA, again comparing different MMLV RT Mutants versus thewild-type enzyme, and AMV RT. The aRNA yields were determined by UVabsorbance at 260 nm and were performed in duplicate. Although theF155Y;H638G MMLV RT produced the most aRNA in this experiment, it isimportant to note that the single mutant H638G MMLV RT produced almostas much aRNA as the F155Y;H638G double mutant, and thus represents anoteworthy improvement over the currently available reversetranscriptase tools.

Comparable 2- to 4-fold enhancements in aRNA yield by F155Y;H638G MMLVRT were observed in two round RNA amplification reactions, starting with1 ng, 100 pg, or 10 pg total HeLa-S3 RNA. FIG. 8 is a graph that shows atwo round RNA amplification with 10-1000 pg HeLa S-3 Total RNA, againcomparing different MMLV RT Mutants versus wild-type. AMV=AvianMyeloblastosis Virus RT; FYHG=F155Y;H638G MMLV RT; SSII=SuperScript II.The aRNA yields were determined by UV absorbance at 260 nm and wereperformed in duplicate.

In another study, the aRNA yield by F155Y;H638G MMLV RT was compared inMessageAMP with AMV RT using 100 ng and 1 ug input total RNA. As shownin Table 6, F155Y;H638G MMLV RT produced 12% more aRNA from 1 ug oftotal RNA, and 2.6-fold more aRNA from 100 ng total RNA (both at 200 UF155Y;H638G MMLV RT).

TABLE 5 Yields of aRNA by F155Y; H638G MMLV RT compared to AMV RT at 100ng and 1 ug of HeLa-S3 Total RNA. RT, Units 1 ug 100 ng F155Y; H638G,100 U 56.2 6.1 F155Y; H638G, 100 U 54.1 6.6 F155Y; H638G, 100 U 54.0 7.7F155Y; H638G, 200 U 66.6 17.6 F155Y; H638G, 200 U 65.8 16.2 F155Y;H638G, 200 U 59.6 16.5 Wild-type AMV 56.8 6.2 Wild-type AMV 57.9 6.5

In another study, the aRNA yield by F155Y;H638G MMLV RT was comparedwith AMV RT in a two round amplification using MessageAMP using 10 pginput total HeLa-S3 RNA. FIG. 9 is graph that shows the yield from a tworound RNA amplification comparing F155Y;H638G MMLV RT and AMV RT. Theinput total RNA was HeLa-S3, at 1 ng and 10 pg. As shown in FIG. 9,F155Y;H638G MMLV RT produced 22-fold more aRNA than the RT provided inthe kit, and nearly 40-fold more aRNA than wild-type AMV RT.

In yet another study, the aRNA yield by F155Y;H638G MMLV RT was comparedwith the Affymetrix aRNA standard protocol which recommends SuperScriptII. In this case, the F155Y;H638G MMLV RT mutant generated 7% more aRNAfrom 1 ug of total RNA (HeLa-S3), or 20% more aRNA from 100 ng of totalRNA, in a single round of RNA amplification. FIG. 10 is a graph thatshows a comparison of the present invention with the Standard AffymetrixaRNA Protocol Comparison to MessageAMP containing F155Y;H638G MMLV RT.An input 1000 and 100 ng of HeLa-S3 total RNA was amplified using theAffymetrix aRNA Protocol or Ambion's MessageAMP protocol usingF155Y;H638G MMLV RT. aRNA labeling was accomplished through 8 hr biotinCTP/UTP IVT reactions. All reactions were performed in duplicate.Average peak sizes of the aRNA exceed 1700 nucleotides in each case.SSII=SuperScript II; DM=Double mutant, F155Y;H638G MMLV RT. EXAMPLE 8

Performance of aRNA produced by F155Y;H638G MMLV RT and SSII onAffymetrix GeneChips. RNA amplified in a single round by F155Y;H638GMMLV RT (using the MessageAMP II protocol (see www.ambion.com)) or theRT provided in the kit (using the Affymetrix aRNA protocol (seewww.Affymetrix.com)) was biotin-labeled and hybridized to an AffymetrixHuman Focus Array for detection and analysis. The concordance betweenthe two RT enzymes was 93.69% using all 8794 elements on the array. FIG.11 is a scatter plot of F155Y;H638G MMLV RT (x axis) vs. SSII (y axis)Signal Intensities from a Human Focus Array. The signal intensitycorrelation is shown. Lines represent 2-, 3-, 10- and 30-folddifferences. Red dots are Present-Present calls, Black are Absent-Absentcalls, and Dark Blue are Present-Absent (or Absent-Present). This isgraph is used to visualize the concordance between arrays. The region onthe top, right half contains the P calls (most important). DM=DoubleMutant, F155Y;H638G MMLV RT.

Elimination of Absent and Marginal calls increases the concordancesubstantially. The average signal was 1348 for F155Y;H638G MMLV RT, and1291 for the Affymetrix standard protocol. Percent present calls wereslightly higher for F155Y;H638G MMLV RT than the standard protocol(Table 6), whereas the beta-actin ratio was also slightly more favorablefor F155Y;H638G MMLV RT than the RT provided in the kit (Table 7).

TABLE 6 Percent Present Calls on the Human Focus Array by RT Enzyme RTEnzyme % Present Calls F155Y; H638G MMLV RT 54.0% SSII 53.8%

TABLE 7 3′/5′ Ratios for GAPDH and beta-Actin Genes on the Human FocusArray by RT Enzyme. SSII F155Y; H638G MMLV RT GAPDH 0.79 0.80 Beta-Actin1.24 1.12

While this invention has been described in reference to illustrativeembodiments, this description is not intended to be construed in alimiting sense. Various modifications and combinations of theillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thedescription. It is therefore intended that the appended claims encompassany such modifications or embodiments.

1-61. (canceled)
 62. An isolated and purified nucleic acid comprising ahyperactive reverse transcriptase with a mutation in the processivitydomain and in the nucleotide selection domain.
 63. The nucleic acid ofclaim 62, wherein the hyperactive reverse transcriptase comprises amutation that corresponds to and includes an H638G mutation of theMMLV-RT.
 64. The nucleic acid of claim 62, wherein the hyperactivereverse transcriptase comprises a hyperactive reverse transcriptasefurther comprising an F155Y mutation.
 65. The nucleic acid of claim 62,wherein the hyperactive reverse transcriptase comprises a hyperactivereverse transcriptase further comprising an F155Y mutation and an H638Gmutation.
 66. The nucleic acid of claim 62, wherein the nucleic acid ofSEQ ID No.: 1 further comprises a nucleic acid segment encoding a leadersequence.
 67. The nucleic acid of claim 62, wherein the nucleic acid ofSEQ ID NO.: 1 further comprises a nucleic acid segment encoding aprotein segment other than the hyperactive reverse transcriptase.
 68. Anisolated and purified nucleic acid that encodes a hyperactive reversetranscriptase comprising one or more mutations in the processivitydomain.
 69. The reverse transcriptase of claim 68, wherein the mutationin the processivity domain comprises one or more of mutations in thefollowing residues in MMLV-RT: H638, Y586, D653, D524, D524 and E562.70. The nucleic acid of claim 68, wherein the hyperactive reversetranscriptase comprises an H638G mutation.
 71. The nucleic acid of claim68, wherein the hyperactive reverse transcriptase comprises ahyperactive reverse transcriptase further comprising an F155Y mutationand an H638G mutation.
 72. The nucleic acid of claim 68, wherein thenucleic acid of SEQ ID NO.: 1 further comprises a nucleic acid segmentencoding a leader sequence.
 73. The nucleic acid of claim 68, whereinthe nucleic acid of SEQ ID NO.: 1 further comprises a nucleic acidsegment encoding a protein segment other than the hyperactive reversetranscriptase. 74-79. (canceled)
 80. A process for making an isolatedhyperactive reverse transcriptase comprising the steps of: transforminga host cell with an isolated nucleic acid that encodes a hyperactivereverse transcriptase; and culturing the host cell under conditions suchthat the hyperactive reverse transcriptase is produced.
 81. The processof claim 80, wherein the hyperactive reverse transcriptase comprises anH638G mutation.
 82. The process of claim 80, wherein the hyperactivereverse transcriptase comprises a hyperactive reverse transcriptasefurther comprising an F155Y mutation.
 83. The process of claim 80,wherein the hyperactive reverse transcriptase comprises a hyperactivereverse transcriptase further comprising an F155Y;H638G mutation.84-128. (canceled)